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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 1, 2004 as doi:10.1096/fj.03-0875fje. |
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Department of Physiology, University of Kentucky, A. B. Chandler Medical Center, Lexington, Kentucky, USA
2Correspondence: Department of Physiology, University of Kentucky, A. B. Chandler Medical Center, MS 508, 800 Rose St., Lexington, KY 40536, USA. E-mail: mnikolo{at}uky.edu
SPECIFIC AIMS
Neutral plasma membrane sphingomyelinase (NSMase) has been suggested to participate in numerous cellular responses, including inflammation, cell growth arrest, and apoptosis. Until recently, however, the proteins responsible for this activity were not purified or sequenced. Based on homology searches with bacterial SMase, a novel putative NSMase (NSMase-2) was identified and characterized. The goal of this study was to test whether NSMase-2 plays a role in signal transduction, particularly in IL-1ß signaling.
PRINCIPAL FINDINGS
1. Molecular cloning and functional characterization of NSMase-2
The I.M.A.G.E consortium clone 2076661, which is identical to the published mouse NSMase-2 cDNA (AJ250461), was used. NSMase-2 was PCR amplified, sequenced, FLAG-tagged at the carboxyl terminus, and cloned into the shuttle vector pAd.trac.teton under doxycycline-inducible promoter. The resulting construct, pAd.trac.teton-NSMase-2, was used for the initial characterization of NSMase-2 in HEK 293 cells. NSMase-2 appeared on a Western blot as a single band of apparent molecular mass of 73 kDa. The induction of expression resulted in a significant increase in the initial velocity of NSMase activity, from 1 pmol/min in the noninduced cells to 30 pmol/min in induced cells. HPLC analyses confirmed that only one product, NBD-ceramide, was generated at the expected molar ratio of 1:1. Addition of 0.5% Triton X-100 or 50 mM EDTA to the assay mixture suppressed the inducible NSMase activity.
Recombinant adenovirus expressing NSMase-2 (AdNSMase-2) was generated and used to infect primary hepatocytes. As in HEK cells, induction of NSMase-2 expression led to the production of a 73 kDa protein with significant NSMase activity. More important, overexpression of NSMase-2 resulted in increases in cellular ceramide levels by 30%, 62%, and 73% at 16, 48, and 72 h. This corresponds to an increase by 0.5, 1.25, and 1.3 nmol/mg protein, respectively, and indicates that the expressed NSMase-2 hydrolyses endogenous substrate and generates ceramide in the cells. No statistically significant differences were found in the viability of hepatocytes among the groups.
2. Subcellular localization of NSMase-2
Neutral sphingomyelinase activity is associated mainly with the plasma membrane, and the predicted amino acid sequence for NSMase-2 reveals two putative transmembrane domains at the amino terminus. Subcellular localization of the FLAG-tagged protein was determined by confocal microscopy using anti-FLAG antibody in Hep G2 cells, showing that NSMase-2 was localized mainly at the plasma membrane (Fig. 1
).
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Taken together, these results indicate that the cloned NSMase-2 encodes a functionally active Mg2+-dependent and plasma membrane-bound NSMase.
3. Effects of NSMase-2 expression on IL-1ß-induced JNK activation
Our previous studies showed that IL-1ß stimulates NSMase activity in primary hepatocytes, so it was of interest to see whether NSMase-2 has a role in IL-1ß signaling. One of the most important targets of IL-1ß is JNK, a member of the MAP kinase family. As anticipated, treatment with IL-1ß increased transiently the level of the active, phosphorylated form of JNK. (Fig. 2
A, B). NSMase-2 overexpression enhanced up to threefold the potential of IL-1ß to induce JNK phosphorylation whereas in the absence of IL-1ß, NSMase-2 overexpression had only a small effect on JNK. A dose response study (Fig. 2C, D
) confirmed that the magnitude of IL-1ß-induced JNK phosphorylation was substantially increased in the presence of NSMase-2 over a broad range of IL-1ß concentrations.
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4. The potentiation effects of NSMase-2 on JNK phosphorylation are not affected by the tyrosine kinase inhibitor AG126
The IL-1ß receptor-associated kinase IRAK has a central role in the IL-1ß signaling cascade. Upon receptor activation, it is hyperphosphorylated by itself and by other kinase(s), such as IRAK-4, which triggers the release of a multiprotein signaling complex and activation of the MAP kinase cascade (Fig. 3
). To test whether NSMase-2 overexpression affects IL-1ß signaling cascade up- or downstream of IRAK, we used AG126. This putative tyrosine phosphorylation inhibitor has been found to prevent lipopolysaccharide-induced IRAK activation. Control experiments showed that AG126 inhibited IL-1ß-induced phosphorylation of IRAK by 50% at most, probably because AG126, a tyrosine kinase inhibitor, cannot block IRAK autophosphorylation that occurs at serine or threonine residues. The addition of AG126 also prevented IL-1ß-induced JNK phosphorylation, but only when NSMase-2 expression was not induced. In contrast, AG 126 had only a partial effect in doxycycline-induced cells, not being able to inhibit the "potentiation" effect of NSMase-2.
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5. NSMase-2 overexpression alters the phosphorylation pattern of IRAK
It is known that hyperphosphorylation of IRAK-1 plays a dual role in IL-1ß signaling. It triggers the release of IRAK-1 from the receptor complex, leading to binding and activation of TAK-1. At the same time, however, phosphorylated amino acids act as targets for IRAK ubiquitination and degradation, which shuts down the signaling pathway. We decided to test in more detail the effect of NSMase-2 on IRAK phosphorylation.
IL-1ß induced IRAK phosphorylation as early as 8 min after treatment, but this effect was significantly suppressed when NSMase-2 was overexpressed, indicating that NSMase-2 overexpression maintained IRAK in a less phosphorylated state. To confirm this, cell lysates were treated with bacteriophage protein phosphatase, which reduced the appearance of high molecular weight forms of IRAK while increasing the intensity of the nonphosphorylated form of IRAK only in control cells (that did not overexpress NSMase-2). These results suggested that NSMase-2 overexpression might inhibit a serine-threonine kinase or activate a protein phosphatase.
6. NSMase-2 potentiates IL-1ß-induced JNK activation in a PP2A-dependent manner
Among the most well-characterized ceramide targets is ceramide-activated protein phosphatase, a member of the PP2A family of protein phosphatases that can be selectively inhibited by 10 nM okadaic acid (OA). We tested whether OA treatment would affect the ability of NSMase-2 to enhance the IL-1ß-induced JNK activation. OA abolished the potentiative effect of NSMase-2, indicating that the ability of NSMase-2 to augment IL-1ß-induced JNK activation indeed is mediated by activation of a member of PP2A family.
CONCLUSIONS AND SIGNIFICANCE
Ceramide is a bioactive second messenger molecule that participates in variety of stress responses. The major pathway for elevating cellular ceramide content is via activation of sphingomyelinase, which catalyzes the turnover of sphingomyelin to phosphorylcholine and ceramide.
This study provides the first evidence that NSMase-2, an enzyme cloned by sequence homology analyses and proposed to be a Mg2+-dependent neutral SMase, can modulate the IL-1ß-induced JNK activation. It also presents data that, in hepatocytes, the enzyme is localized to the plasma membrane and can regulate intracellular ceramide content. Enzymatic analyses of the cloned NSMase-2 confirm the NSMase nature of the enzyme and its Mg2+ dependency.
Experimental data accumulated over the last decade have suggested that bioactive ceramide is mainly synthesized at the plasma membrane, but until now neither of the two putative NSMases had been localized to the plasma membrane. The only study published on the subcellular localization of NSMase-2 reported an exclusive Golgi localization. In contrast, our studies find that, in hepatocytes, NSMase-2 is localized at the plasma membrane. These differences could be due to 1) differences rising from the different cell type or 2) the fact that our studies used antibody against the exogenously expresses NSMase-2, whereas the aforementioned study used antibody against the endogenous enzyme. This finding offers new insight into the possible cellular functions of NSMase-2 and shows that enzyme is in close proximity to key components of the IL1ß signaling cascade such as IL-1ß receptors, IRAK, and the TGFß-activated kinase, TAK-1.
Increases in endogenous ceramide levels by addition of exogenous ceramide analogs or by treatment of cells with bacterial sphingomyelinase have been known to induce JNK phosphorylation in different cell types. Our findings provide the first evidence that endogenous generation of ceramide at the plasma membrane modulates JNK activation, and implicate NSMase-2 in the process. These studies also suggest that the mechanism for the effects of NSMase-2 on JNK phosphorylation does not involve effects on IRAK phosphorylation at tyrosine residues. NSMase-2 overexpression seems to keep IRAK in a less phosphorylated form by activating a serine-threonine phosphatase from the PP2A family.
The mechanism by which dephosphorylation of the hyperphosphorylated IRAK-1 leads to potentiation of JNK activation is unclear. Phosphorylated amino acids in IRAK are targets for ubiquitin ligase. IRAK’s ubiquitination leads to its degradation and shuts down the signaling pathway. It is therefore conceivable that dephosphorylation could prevent IRAK degradation and result in enhanced downstream signaling. Alternatively, since only the nonphosphorylated IRAK interacts with MyD88, dephosphorylation may increase IRAK’s affinity for MyD88 and anchor the kinase back to the receptor, leading to a more sustained activation of the cascade. Our ongoing studies are aimed at testing these possibilities.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0875fje; 
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B. The signaling cascade is shut down by ubiquitination and degradation of hyperphosphorylated IRAK. The putative Mg2+-dependent neutral SMase, NSMase-2, is localized at the plasma membrane, generates excess ceramide, and activates a serine threonine phosphatase from the PP2A family. This keeps IRAK in a less phosphorylated form and potentiates the activation of JNK by a mechanism not yet fully understood.